The present invention relates to a structured illumination microscope device and an image forming apparatus.
In the field of observation and measurement of the micro-structures of samples, observation at higher spatial resolution is demanded. As a method to improve the lateral resolution of a sample, a method of modulating a sample with structured lights during photographing and demodulating the captured image by image processing is known as “structured illumination microscopy”, and is disclosed in U.S. Pat. No. 6,239,909 and U.S. Pat. No. RE38,307, for example.
The sixth embodiment disclosed in U.S. Pat. No. 6,239,909 is an example when the structured illumination microscope apparatus is applied to a fluorescence microscope, and the optical system thereof splits the illumination light emitted from a coherent light source using such beam splitting means as a diffraction grating, then condenses the illumination beams into a pupil plane of an objective lens, and emits the illumination beams as parallel beams from the objective lens at different angles, so as to overlap around the observation object and form an interference fringe.
Because the illumination light is modulated into a fringe, diffracted lights containing spatial frequency components of the shape information of the observation object, which cannot be transferred by a conventional imaging system, can be used for image formation. And by relatively modulating the phase of the split illumination beams and moving the interference fringe on the observation object when a plurality of images are acquired, image formation based on image computing processing is enabled.
In concrete terms, phase modulation is performed by moving the diffraction grating vertically to the optical axis, or in another example, by inserting a wedge prism into one of the illumination paths, and moving this wedge prism in a direction vertical to the optical axis.
According to the method disclosed in U.S. Pat. No. RE38,307, illumination light from the coherent light source is guided using an optical fiber, and is split by such beam splitting means as a diffraction grating, and then the illumination beams are condensed into a pupil plane of the objective lens so as to form an interference fringe near the observation object. Because the illumination light is modulated into a fringe, the high frequency components of the shape information of the observation object, which cannot be transferred by a conventional imaging system, can be used for image formation. And a plurality of images are captured in the same manner, and image formation based on image computing processing is performed.
According to this method, in order to create one image, not only a plurality of images are acquired with phase-modulated structured illumination, but also images are acquired with changing the direction of the structured illumination. This is because the high frequency components can be used for image formation only when the structure has the same direction as the direction of the structured illumination, so in order to restore the shape of the sample which extends two-dimensionally, a plurality of images must be acquired with changing the direction of the structured illumination, and be combined.
In this structured illumination, it is normally desirable that the beams with respect to the interference surface have S polarization in order to allow two or more beams to interfere. Because the contrast of the interference fringe when beams with S polarization is 1, regardless of the incident angle, the contrast when beams enter in the P polarization state attenuates in proportion to cos (Δθ), where Δθ is the crossing angles of the beams. Since the observed light is the sum of S polarization and P polarization, contrast to be observed decreases as P polarization attenuates. At Δθ>90°, the numeric value of the contrast of P polarization becomes negative, which means that the brightness/darkness of the interference fringe inverts, and the interference fringe generated by S polarization is cancelled, which is not desirable.
In particular, the structured illumination microscope is a technology used to obtain high resolution, and it is preferable that the NA of the objective lens to be used is as high as possible, and the pitch of the structured illumination is as short as possible. As a result, the beams for the structured illumination enter into the sample at a large angle, so if a P polarization component exists, attenuation of the P polarization is major since the above mentioned Δθ is large, which becomes the cause of deterioration of the structured illumination contrast.
In order to address this problem, a non-polarization state is created by allowing light from the light source to pass through a diffuser or vibrating optical fibers, then light is nearly linear polarized using a polarizer, which is installed near the diffraction grating and which rotates coaxially with the diffraction grating, so as to enter with S polarization onto the sample.
As a result, ideal structured illumination contrast is obtained, but the polarizer blocks about half of the light power, which lowers utilization efficiency of the light to 50%.